With the recent completion of the human genome sequence, the sequencing of other vertebrate genomes has taken center stage. The sequencing of widely studied model organisms (e.g., mouse, rat, and zebrafish) is providing a valuable resource for future experimentation and important insights into vertebrate biology. Less clear is the relative value of other candidate genomes being considered for systematic sequencing, especially with regard to their potential contribution to the annotation and interpretation of the human genome sequence. To investigate such issues, we are generating large blocks of orthologous sequence from multiple vertebrates for detailed comparative analyses. Specifically, the same targeted genomic regions from multiple vertebrate specie are being isolated in large-insert clones and then sequenced. Efficient methods for designing orthologous hybridization probes and isolating bacterial artificial chromosome (BAC) clones from the different species have been developed and implemented. Following characterization by several mapping methods, tiling paths of BACs are then selected and systematically sequenced. In total, 250 Mb of comparative sequencing data is being generated each year, in conjunction with the Physical Mapping Section of the NHGRI Genome Technology Branch. The establishment of this comparative sequence resource is facilitating the development of new computational tools for multi-species sequence comparisons, providing insight about the appropriate degrees of sequencing finishing that should be pursued in the sequencing of other vertebrate species, and revealing the benefits of sequencing species from a range of different evolutionary distances from human. These efforts are being extensively focused on the ENCODE project, which aims to identify all functional in the human genome. Indeed, the great majority of NISC sequencing over the past year has been dedicated to the ENCODE project. In addition to its inter-species sequence comparisons, NISC has developed a second major sequence-production pipeline designed for performing intra-species (specifically, inter-human) sequence comparisons for medical research projects. In establishing and utilizing a PCR-based sequencing pipeline, NISC is once again capitalizing on its unique circumstance of being embedded within the broader NIH Intramural Program with its outstanding clinical research infrastructure. Among the currently planned NISC medical sequencing projects is a large effort (ClinSeq) that directly interfaces with well-established clinical researchers at other NIH Institutes and utilizes the NIH Clinical Center to study the molecular basis for common human diseases, with an emphasis on the detection and study of rare disease-associated variants. Together, the two pipelines of the NISC Comparative Sequencing Program should continue to produce data at the cutting edge of genomics research, exploring how large-scale DNA sequencing can be used to characterize the human genome and to understand the genetic basis for human health and disease. In addition, NISC has recently launched a major effort to characterize the microbes living on human skin-- the skin microbiome. Understanding the collection of microbes, the great majority of which cannot be cultured in the laboratory, is critical for understanding their role in human health and disease. With a focus on the skin, NISC aims to characterize the human skin microbiome as part of the Human Microbiome Project. Finally, NISC is heavily engaged in implementing the powerful new 'next-generation'sequencing technologies into its pipelines. Extensive testing of available platforms has been ongoing, providing important insights about which new technologies are best-suited for full-scale implementation at NISC. Virtually all sequencing projects will be affected by the implementation of these new technologies.